DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.     

A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato.

The elicitor-induced activation of the potato pathogenesis-related gene PR-10a is positively controlled by a protein kinase(s) that affects the binding of the nuclear factors PBF-1 (for PR-10a binding factor-1) and PBR-2 to an elicitor response element (ERE). In this study, we have identified a kinase that has properties similar to the conventional isoenzymes of the mammalian protein kinase C (PKC) family. the treatment of potato tuber discs with specific inhibitors of PKC abolished the elicitor-induced binding of the nuclear factor PBF-2 to the ERE. This correlated with a reduction in the accumulation of the PR-10a protein. In contrast, treatment of the tuber discs with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, led to an increase in binding of PBF-2 to the ERE and the corresponding increase in the level of the PR-10a protein, mimicking the effect seen with the elicitor arachidonic acid. Biochemical characterization of proteins extracted from the particulate fraction of potato tubers demonstrated that a kinase belonging to the conventional isoforms of PKC is present. This was confirmed by immunoprecipitation with antibodies specific to the conventional isoforms of human PKC and in-gel kinase assays. The ability of the immunoprecipitates to phosphorylate the alpha-peptide (a PKC specific substrate) in the presence of the coactivators calcium, phosphatidylserine, and TPA strongly suggested that the immunoprecipitated kinase is similar to the kinase characterized biochemically. Finally, the similar effects of the various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato.     

Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein

The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).     

The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.     

The DNA binding mechanism of a SSB protein from Lactococcus lactis siphophage p2

Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.     

Structure of the full-length human RPA14/32 complex gives insights into the mechanism of DNA binding and complex formation

Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1-42) and C-terminal (residues 175-270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.     

The cross-reactive calcium-binding pollen allergen,Phl p 7, reveals a novel dimer assembly

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.     

A distinctive single-strand DNA-binding protein from the Archaeon Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryarchaeal counterparts. Rather than comprising four DNA-binding domains and a zinc-finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus SSB protein (SsoSSB) has a single DNA-binding domain in a polypeptide of just 148 amino acids with a eubacterial-like acidic C-terminus. SsoSSB protein was purified to homogeneity and found to form tetramers in solution, suggesting a quaternary structure analogous to that of E. coli SSB protein,despite possessing DNA-binding domains more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately five nucleotides (nt)per monomer. Additionally, the protein is functional both in vitro and in vivo, stimulating RecA protein-mediated DNA strand-exchange and rescuing the ssb-1 lethal mutation of E. coli respectively. We discuss possible evolutionary relationships amongst the various members of the SSB/RPA family.     

The PB1 domain and the PC motif-containing region are structurally similar protein binding modules

The PC motif is evolutionarily conserved together with the PB1 domain, a binding partner of the PC motif-containing protein. For interaction with the PB1 domain, the PC motif-containing region (PCCR) comprising the PC motif and its flanking regions is required. Because the PB1 domain and the PCCR are novel binding modules found in a variety of signaling proteins, their structural and functional characterization is crucial. Bem1p and Cdc24p interact through the PB1-PCCR interaction and regulate cell polarization in budding yeast. Here, we determined a tertiary structure of the PCCR of Cdc24p by NMR. The tertiary structure of the PCCR is similar to that of the PB1 domain of Bem1p, which is classified into a ubiquitin fold. The PC motif portion takes a compact betabetaalpha-fold, presented on the ubiquitin scaffold. Mutational studies indicate that the PB1-PCCR interaction is mainly electrostatic. Based on the structural information, we group the PB1 domains and the PCCRs into a novel family, named the PB1 family. Thus, the PB1 family proteins form a specific dimer with each other.     

VirE2: a unique ssDNA-compacting molecular machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.     

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.     

Characterization of the single stranded DNA binding protein SsbB encoded in the Gonoccocal Genetic Island

Background

Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins.

Methodology/Principal Findings

In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg²⁺ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity.

Conclusions/Significance

We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands.     

Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing

Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.     

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D₁ ~ 5000 nt² s^− 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.